The following work was accomplished in 2007:[unreadable] [unreadable] Expression, Purification and Complexation of Bacterial and Human Proteins:[unreadable] [unreadable] An expression system for TbpA has been developed and is capable of producing 0.2-0.4 mg/L of purified protein. A large scale preparation (27L) of E. coli transformed with a plasmid containing the gene for TbpA as well as an N-terminal His-Tag and a cleavage site are grown and induced with low level IPTG overnight and then harvested. The cells are then lysed and membranes are separated via ultracentrifugation, and the membrane proteins are then solubilized in detergent. This protein mixture is run over a Nickel-NTA column. The Nickel eluate is then further purified (and detergent exchanged if necessary) via gel filtration chromatography. This purified protein can also be complexed with purified commercially available iron-free or iron-bound human serum transferrin. Crystallization trials are underway.[unreadable] [unreadable] An expression system for TbpB has also been developed and is capable of producing 7-8 mg/L of purified protein. A medium scale preparation (4L) of E. coli transformed with a plasmid containing the gene for TbpB (a soluble construct lacking the N-terminal membrane anchor) as well as an N-terminal His-Tag and a cleavage site are grown and induced with high level IPTG for a few hours and then harvested. The cells are then lysed and membranes are separated via ultracentrifugation, and the soluble fraction is run over a Nickel-NTA column. The Nickel eluate is then further purified and can be bound to purified commercially available iron-bound human serum transferrin. Furthermore, purification of a triple complex of TbpA, TbpB and transferrin is in progress. Crystallization trials of the complexes are underway.[unreadable] [unreadable] Structure Determination of Iron-Free Human Serum Transferrin:[unreadable] [unreadable] We recently solved the 2.7 A crystal structure of iron-free human transferrin using X-ray diffraction and molecular replacement phasing. This structure shows several interesting features that might be important not only for iron-binding, but also for the receptor binding properties of transferrin. The structure resembles that of other serum transferrins, having two similar lobes with the iron-coordinating residues in a similar arrangement. However, in the human serum transferrin structure, the lobes are almost equally open, and a citrate molecule (a component of the crystallization buffer) is found within the iron binding region. Citrate is also found to form a crystal contact between the transferrin molecules in the crystal. An interesting feature of the structure is a difference in the hinge region of each lobe which might influence their differing iron affinities. A comparison of the crystal structure of human serum transferrin with human lactoferrin shows several important differences, including an overall difference in the relative orientations of the two lobes of the proteins. This difference between human serum transferrin and lactoferrin might be due to structural differences between the inter-lobe linkers, as well as differences in the pattern of disulfide bonding and the nature of the polar contacts between the lobes. These features may be important for the different receptor binding and iron-binding properties of these proteins.